Aim

This module consists on all the necessary tests to prove the efficiency of the effectors that the bacteriophage will introduce in the cancerous cells. First, apoptin will trigger apoptosis in cancer cells. Furthermore, by using siRNA designed for survivin, the rate of division of cancer cells will be decreased to turn them more vulnerable to apoptin. Thus, the combination of both effectors will induce apoptosis more strongly.

Background

Apoptosis

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“Apoptosis (or programmed cell death) is an active and programmed physiological process for eliminating superfluous, altered, or malignant cells.” [1]. One of the mechanisms by which cancer cells are erradicated, it is an evolutionary conserved, intrinsic program of cell death that occurs in various physiological and pathological situations. It is characterized by typical morphological and biochemical hallmarks, including cell shrinkage, nuclear DNA fragmentation and membrane blebbing. In addition, proteolytic enzymes such as caspases are important effector molecules in apoptosis. Activation of caspases can be initiated from different entry points, for example, at the plasma membrane upon binding of a death receptor (receptor pathway) or at the mitochondria (mitochondrial pathway). The receptor signaling pathway can principally be inhibited by an increase in antiapoptotic molecules or by a decrease or defective function in proapoptotic proteins. (Fulda et al., 2006)

The TP53 gene, which encodes p53, is one of the most frequently mutated genes in human cancers. More than 26,000 somatic mutation data of p53 appear in the international agency for research on cancer. It is reported that approximately half of all cancers have inactivated p53. The p53 protein has a broad range of biological functions, including regulation of the cell cycle, apoptosis, senescence, DNA metabolism, angiogenesis, cellular differentiation, and the immune response. (Suzuki et al., 2011) The effectors used in this therapy are p53 independent, so it is less likely that they will interfere in non-cancerous cells.

<figure> <a href="" data-lightbox="p53_Pathway" data-title="Fig. 4.1: Apoptosis pathways taken from www.sinobiological.com"><img class="img img-responsive" style="margin:0px auto;display:block; width:360px;" src=""></a>

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Survivin siRNA

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“Survivin, a member of the inhibitor-of-apoptosis gene family, is a multifunctional protein that suppresses apoptosis and regulates progression through the G2/M phase.” (Kappler et al., 2004) By applying small interfering RNA (siRNA) to the cells, gene transcription can be silenced. Transcription silencing is thought to be a defense strategy that preserves genomes through evolution from attack by double-stranded RNA viruses and transposing [2].

SiRNA works by promoting RNA degradation in a highly sequence-specific manner. This means siRNA needs to be complementary to the messenger RNA (mRNA) it wants to silence. With longer interfering RNA, and in general, double stranded RNA, the endoribonuclease Dicer acts to randomly fragment it into siRNA. Next, RNA-Induced Silencing Complex (RISC) takes up siRNA and uses one strand of it to recognize the complementary mRNA and cleave it, decreasing the available mRNA to code for a specific protein, thus silencing or decreasing its expression. [4]

As survivin is a highly expressed protein in cancer cells and embryonic tissue, treatment of cancer with either survivin-specific antisense oligonucleotides or ribozymes has been done and resulted in reduced survivin expression, which has been correlated with a decrease in tumor growth in mice, with an increase in apoptosis, and with the formation of polyploid cells in vitro. [2]

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Apoptin

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“Apoptin is a small protein produced by Chicken Anemia Virus (CAV) that is capable of inducing apoptosis in human tumor cells while leaving normal cells intact.” [3] “Apoptin-mediated cell death is independent of death receptors such as FADD (Fas dependent death) or caspase-8” [4].

Even though the mechanism remains ambiguously understood, apoptin selectively induces apoptosis in transformed avian or mammalian cell lines, but not in primary, non-transformed cells. In primary cells, apoptin remains in the cytoplasm, whereas in cancer cells it migrates into the nucleus and ultimately kills the cell by the activation of the mitochondrial death pathway, in a Nur77-dependent manner [5]. In tumor cells apoptin is phosphorylated on T108, a modification that is not observed in normal cells, which might be the tag given by the cell to relocate it to the nucleus and for it not to go through proteosomal degradation [4] According to Maddika et al. (2005) apoptin associates with the anaphase-promoting complex, and induces G2/M arrest and apoptosis.

Apoptin has been used as a therapy in vitro and in vivo by Shoae-Hassani et al. (2013) with λ phage nanobioparticles to trigger apoptosis in human breast carcinoma cells, resulting in a safe delivery method without specific targeting to cells and in apoptosis of carcinoma cells.

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Process description

<section id="column"> <figure> <a href="" data-lightbox="Module4" data-title="Fig. 4.2: Action of the effectors of our therapy."><img class="img img-responsive" style="margin:0px auto;display:block; width:400px;" src=""></a>

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The effectiveness of apoptin and survivin knockdown will be evaluated by making three different systems, one with apoptin alone, another one with survivin siRNA, and the third one with apoptin and survivin siRNA. Survivin siRNA will induce cell arrest, therefore its rate of division will decrease which could induce apoptosis in cancer cells [8]. Apoptin is also another pro-apoptotic protein, so by producing it in cancer cells it will induce apoptosis in a p53-independent manner and its tumoricidal activity is not affected by the overexpression of B-cell lymphoma 2 (Bcl-2) which is important as the mutation of p53 and Bcl-2 overexpression in tumor cells are mechanisms for chemotherapeutic drug resistance [7]. By combining apoptin and survivin knockdown, it is expected a major apoptotic rate, therefore, more effectiveness as an anti-cancer therapy.

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Biobricks

The next section describes the different biobricks in which our effectors were placed, which were designed to be ligated with the <a href="#">phagemid </a>.

C4 (GFP) (<a href=" http://parts.igem.org/Part:BBa_K1366106" target="_blank">BBa_K1366106</a>)

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This construct will be used as a proof of concept to demonstrate that the phage is capable of transfecting DNA to cancerous cells, and, at the same time, these cells are able to express the phagemid.

<figure> <a href="" data-lightbox="hTERT-GFP" data-title="Fig. 4.3: Structure of construct 4."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src=""></a>

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C5 Apoptin(<a href=" http://parts.igem.org/Part:BBa_K1366104" target="_blank">BBa_K1366104</a>)

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The 13kDa protein Apoptin gets phosphorilated in treonin 108 only in cancerous cells, which enables it to be translocated to the nucleous and subsequently trigger apoptosis. This effector will be expressed under a CMV constitutive promoter because of the high selectivity of the protein to cancer cells.

<figure> <a href="" data-lightbox="CMV-apoptina" data-title="Fig. 4.4: Structure of construct 3."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src=""></a>

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Survivin siRNA

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Small interfering RNA (siRNA) is used to decrease or stop the rate of expression of an specific protein pretranslationally. In this case, it wil be used to decrease survivin’s expression, which is an overexpressed protein in many cancer cell lines that has the ability to bind to some caspases, which inhibits their activity and prevents apoptosis initiation. By inhibiting survivin expression, cell arrest and apoptosis of cancerous cells are expected. Two siRNAs found in literature and one designed by us are used to observe their differences. These constructs are expressed with hTERT promoter, which only activates in cancerous cell lines that have telomerase overexpression.

C6 (siRNA1)(<a href="http://parts.igem.org/Part:BBa_K1366105" target="_blank">BBa_K1366105</a>)

<figure> <a href="" data-lightbox="siRNA1" data-title="Fig. 4.5: Structure of construct 6."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src=""></a>

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C7 (siRNA2)(<a href=" http://parts.igem.org/Part:BBa_K1366106" "target="_blank">BBa_K1366106</a>)

<figure> <a href="" data-lightbox="siRNA2" data-title="Fig. 4.6: Structure of construct 7."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src=""></a>

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C8 (siRNA3)(<a href=" http://parts.igem.org/Part:BBa_K1366107" "target="_blank">BBa_K1366107</a> target="_blank")

<figure> <a href="" data-lightbox="siRNA3" data-title="Fig. 4.7: Structure of construct 8."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src=""></a>

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C9 (Apoptin - siRNA)(<a href=" http://parts.igem.org/Part:BBa_K1366108" "target="_blank">BBa_K1366108</a>")

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At last, both effectors were included in the same construct, for us to observe if there is synergy, antagonism, or have no effect against each other.

<figure> <a href="" data-lightbox="Apoptin - siRNA" data-title="Fig. 4.8: Structure of construct 9."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src=""></a>

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Apoptin siRNA History

To see the graphical Apoptin siRNA History <a href="images/module4/pET_apoptin_siRNA_History.png" data-lightbox="Graphical Summary" data-title="Fig. 4.9: Apoptin siRNA History.">Click here</a>.

Results

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References

1. Danen-Van Oorschot, A., Klein, B., Zhuang, S., Van Der Eb, A., Noteborn, M., Fischer, D., & ... Falkenburg, J. (1997). Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proceedings Of The National Academy Of Sciences Of The United States Of America, 94(11), 5843-5847. doi:10.1073/pnas.94.11.5843

2. Kappler, M., Bartel, F., Panian, M., Schmidt, H., Taubert, H., Bache, M., & ... Meye, A. (2004). Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53. Cancer Gene Therapy, 11(3), 186-193. doi:10.1038/sj.cgt.7700677

3. Lanz, H. L., Suijker, J. J., Noteborn, M. M., & Backendorf, C. C. (2012). Proteasomal insensitivity of apoptin in tumor cells. Biochemical And Biophysical Research Communications, 422(1), 169-173. doi:10.1016/j.bbrc.2012.04.132

4. Lee, S., Son, S., Yhee, J., Choi, K., Kwon, I., Kim, S., & Kim, K. (2013). Structural modification of siRNA for efficient gene silencing. Biotechnology Advances, 31(5), 491-503. doi:10.1016/j.biotechadv.2012.09.002

5. Maddika, S., Booy, E. P., Johar, D., Gibson, S. B., Ghavami, S., & Los, M. (2005). Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway. Journal of cell science, 118(19), 4485-4493.

6. Shoae-Hassani, A., Keyhanvar, P., Seifalian, A., Mortazavi-Tabatabaei, S., Ghaderi, N., Issazadeh, K., & ... Verdi, J. (2013). λ Phage Nanobioparticle Expressing Apoptin Efficiently Suppress Human Breast Carcinoma Tumor Growth In Vivo. Plos ONE, 8(11), 1-12. doi:10.1371/journal.pone.0079907

7. Yuan, L., Zhang, L., Dong, X., Han, D., Liu, X., Li, S., & Zhao, H. (2012). Apoptin selectively induces the apoptosis of tumor cells by suppressing the transcription of HSP70. Tumor Biology, 34(1), 577-585. doi:10.1007/s13277-012-0585-y

8. Wenying, Z., Zhaoning, J., Zhimin, Y., Dongyun, C., & Lili, S. (2012). Survivin siRNA inhibits gastric cancer in nude mice. Cell Biochemistry And Biophysics, 62(2), 337-341. doi:10.1007/s12013-011-9315-0